The present invention relates to therapeutic compositions of macrophage derived chemokine (MDC) and methods for treating and preventing infection by a lentivirus, in particular an immunodeficiency virus, particularly HIV infection, using MDC proteins, nucleic acids and/or derivatives or analogues thereof. The present invention further relates to methods of prognosis for a lentivirus infection, particularly an HIV infection using the MDC as a prognostic indicator.
Human immunodeficiency virus (HIV) induces a persistent and progressive infection leading, in the vast majority of cases, to the development of the acquired immunodeficiency syndrome (AIDS) (Barre-Sinoussi et al., 1983, Science 220:868-870; Gallo et al., 1984, Science 224:500-503). There are at least two distinct types of HIV: HIV-1 (Barre-Sinoussi et al., 1983, Science 220:868-870; Gallo et al., 1984, Science 224:500-503) and HIV-2 (Clavel et al., 1986, Science 233:343-346; Guyader et al., 1987, Nature 326:662-669). In humans, HIV replication occurs prominently in CD4+ T lymphocyte populations, and HIV infection leads to depletion of this cell type and eventually to immune incompetence, opportunistic infections, neurological dysfunctions, neoplastic growth, and ultimately death.
HIV is a member of the lentivirus family of retroviruses (Teich et al., 1984, RNA Tumor Viruses, Weiss et al., eds., CSH-press, pp. 949-956). Retroviruses are small enveloped viruses that contain a single-stranded RNA genome, and replicate via a DNA intermediate produced by a virally-encoded reverse transcriptase, an RNA-dependent DNA polymerase (Varmus, H., 1988, Science 240:1427-1439). Other retroviruses include, for example, oncogenic viruses such as human T-cell leukemia viruses (HTLV-1, -II,-III), and feline leukemia virus.
Lentiviruses are a subfamily of the retroviruses which include the classic ungulate lentiviruses (visna virus of sheep, caprine arthritis encephalitis virus and equine infectious anemia virus) and the immunodeficiency viruses of humans (HIV), monkeys (SIV), cats (FIV) and cattle (BIV).
Lentivirus particles are approximately 80-110 nm in size and consist of an RNA genome and viral enzymes enclosed in a core of viral proteins encased by a cell-derived membrane spiked with viral envelope glycoproteins. Lentiviruses can be distinguished from other subgroups of retroviruses by a cylindrical or rod-shaped nucleoid in mature particles and the absence of preformed particles in the cytoplasm. Lentiviruses are not oncogenic, but produce long-term, persistent infections which eventually lead to chronic debilitating disease. All lentiviruses studied to date replicate and persist in cells of the monocyte/macrophage lineage. See Encyclopedia of Virology, Vol. 3, page 1316 (Academic Press Limited, London, England, 1994).
The HIV viral particle consists of a viral core, composed in part of capsid proteins designated p24 and p17, together with the viral RNA genome and those enzymes required for replicative events. Myristylated gag protein forms an outer viral shell around the viral core, which is, in turn, surrounded by a lipid membrane envelope derived from the infected cell membrane. The HIV envelope surface glycoproteins are synthesized as a single 160 kilodalton precursor protein which is cleaved by a cellular protease during viral budding into two glycoproteins, gp41 and gp120. gp41 is a transmembrane glycoprotein and gp120 is an extracellular glycoprotein which remains non-covalently associated with gp41, possibly in a trimeric or multimeric form (Hammerskjold, M. and Rekosh, D., 1989, Biochem. Biophys. Acta 989:269-280).
HIV, like other enveloped viruses, introduces viral genetic material into the host cell through a viral-envelope mediated fusion of viral and target membranes. HIV is targeted to CD4+ cells because a CD4 cell surface protein (CD4) acts as the cellular receptor for the HIV-1 virus (Dalgleish et al., 1984, Nature 312:763-767; Klatzmann et al., 1984, Nature 312:767-768; Maddon et al., 1986, Cell 47:333-348). Viral entry into cells is dependent upon gp120 binding the cellular CD4 receptor molecules (Pal et al., 1993, Virology 194:833-837; McDougal et al., 1986, Science 231:382-385; Maddon et al., 1986, Cell 47:333-348), explaining HIV""s tropism for CD4+ cells, while gp41 anchors the envelope glycoprotein complex in the viral membrane. The binding of gp120 to CD4 induces conformational changes in the viral glycoproteins, but this binding alone is insufficient to lead to infection (reviewed by Sattentau and Moore, 1993, Philos. Trans. R. Soc. London (Biol.) 342:59-66).
Studies of HIV-1 isolates have revealed a heterogeneity in their ability to infect different human cell types (reviewed by Miedema et al., 1994, Immunol. Rev. 140:35-72). The majority of extensively passaged laboratory strains of HIV-1 readily infect cultured T cell lines and primary T lymphocytes, but not primary monocytes or macrophages. These strains are termed T-tropic. T-tropic HIV-1 strains are more likely to be found in HIV-1 infected individuals during the late stages of infection (Weiss et al., 1996, Science 272:1885-1886). The majority of primary HIV-1 isolates (i.e., viruses not extensively passaged in culture) replicate efficiently in primary lymphocytes, monocytes and macrophages, but grow poorly in established T cell lines. These isolates have been termed M-tropic. The viral determinant of T- and M- tropism maps to alterations in the third variable region of gp120 (the V3 loop) (Choe et al., 1996, Cell 85:1135-1148; Cheng-Mayer et al., 1991, J. Virol. 65:6931-6941; Hwang et al., 1991, Science 253:71-74; Kim et al., 1995, J. Virol. 69:1755-1761; and O""Brien et al., 1990, Nature 348:69-73). The characterization of HIV isolates with distinct tropisms taken together with the observation that binding to the CD4 cell surface protein alone is insufficient to lead to infection, suggest that cell-type specific cofactors might be required in addition to CD4 for HIV-1 entry into the host cell.
The chemokine receptor CCR5 is normally present in cells of the host and serves as the natural receptor for the xcex2 cysteine-cysteine chemokines RANTES, MIP-1xcex1, and MIP-1xcex2. It serves in addition, however, as a co-receptor for HIV-1 (Feng et al., Science 272:872-877; Cocchi et al., Science 270:1811-1815.)
CCR5 is a seven transmembrane domain, G-protein-coupled protein that is expressed on the surfaces of CD4+ and CD8+ T lymphocytes (types of human peripheral blood mononuclear cells (PBMC) (Raport et al. 1996, J. Biol. Chem. 271:17161-17166), and on KG-1A promyeloblastic cells (Samson et al., 1996, Biochemistry 35:3362-3367). The binding of xcex2 cysteine-cysteine chemokines to CCR5 to these cells triggers a variety of normal cellular events in leukocytes (white blood cells) including increases in intracellular calcium, tyrosine kinase activity and chemotaxis towards areas of inflammation.
In addition to serving these normal signal-transduction and immune response functions, CCR5 serves as a co-receptor that facilitates the attachment and fusion of certain primary M-tropic, non-syncytium inducing strains of HIV-1, to their target cells, i.e., monocyte-macrophages and primary CD4+ T lymphocytes.
Because the chemokines RANTES, MIP-1xcex1 and MIP-1xcex2 compete with HIV-1 for binding to CCR5, they act as natural suppressors of HIV-1 infection (Cocchi et al., 1995, Science 270:1811-1815; Baier et al. 1995, Nature 378:563) and may be part of the body""s general defenses against many types of viruses. Cocchi et al. (1995, Science 270:1811-1815) have shown that RANTES, MIP-1xcex1 and MIP-1xcex2 inhibit infection of monocyte-macrophages and CD4+ T cells by M-tropic HIV-1. They inhibit HIV infection or replication at a stage prior to HIV transcription.
Recently, certain factors produced by activated CD8+ T cells have been implicated in suppression of HIV infection (Walker et al., 1986, Science 234:1563; Brinchman et al., 1990, J. Immunol. 144:2961). The production of a suppressive activity correlates with immune status and shows a steady decline in parallel with HIV disease progression (Walker et al., Cell Immunol. 119:470; Mackewicz et al., J. clin. Invest. 87:1462; Blackbourn et al., 1996, Proc. Natl. Acad. Sci. USA 93:13125). The chemokines RANTES (regulated on activation normal T cell expressed and secreted), macrophage-inflammatory protein-1xcex1 and -1xcex2 (MIP-1xcex1 and MIP-1xcex2, respectively), which are secreted by CD8+ T cells, were shown to suppress HIV-1 p24 antigen production in cells infected with HIV-1, HIV-2 or SIV isolates in vitro (Cocchi et al., 1995, Science 270:1811-1815). Additionally, high levels of these chemokines have been found to be secreted by CD4+ T lymphocytes in individuals that have been exposed to HIV-1 on multiple occasions, but remain uninfected (Paxton et al., 1996, Nature Med. 2:412-417).
However, experiments using acute and endogenous infectivity assays with either primary T cells (Cocchi et al., 1996, Science 270:1811; Barker et al., 1996, J. Immunol. 156:4476; Paliard et al., 1996, AIDS 10:1317; Kinter et al., 1996, Proc. Natl. Acad. Sci. USA 93:14076; Rubbert et al., 1997, AIDS 13:63) or macrophages (Moriuchi et al., 1996, Proc. Natl. Acad. Sci. USA 93:15431) as target cells suggest that the full complement of suppressive activity produced by primary CD8+ T cells is not entirely explained by these chemokines. While RANTES, MIP-1xcex1 and MIP-1xcex2 alone or in combination, potently suppress a variety of primary HIV-1 isolates and macrophage tropic isolates, such as HIV-1BaL, some established laboratory strains, such as HIV-1IIIB, are refractory to inhibition of infection or replication by these chemokines (Cocchi et al., 1995, Science 270:1811-1815). Levels of RANTES, MIP-1xcex1 and MIP-1xcex2 do not correlate with the suppression of certain T-tropic, syncytium-inducing (SI) and T cell line adapted (TCLA) isolates and the addition of neutralizing anti-chemokine antibodies does not reverse the suppressive effect (Barker et al., 1996, J. Immunol. 156:4476; Paliard et al., 1996, AIDS 10:1317; Kinter et al., 1996, Proc. Natl. Acad. Sci. USA 93:14076; Rubbert et al., 1997, AIDS 13:63). Therefore, additional, unidentified chemokines capable of suppressing HIV-1 are produced by activated T cells.
Chemokines, or chemoattractant cytokines, are a subgroup of immune factors that have been shown to mediate chemotactic and other pro-inflammatory phenomena (See, Schall, 1991, Cytokine 3:165-183). Chemokines are small molecules of approximately 70-80 residues in length and can generally be divided into two subgroups, xcex1 which have two N-terminal cysteines separated by a single amino acid (CxC) and xcex2 which have two adjacent cysteines at the N terminus (CC). RANTES, MIP-1xcex1 and MIP-1xcex2 are members of the xcex2 subgroup (reviewed by Horuk, R., 1994, Trends Pharmacol. Sci. 15:159-165; Murphy, P. M., 1994, Annu. Rev. Immunol. 12:593-633). The amino terminus of the xcex2 chemokines RANTES, MCP-1, and MCP-3 have been implicated in the mediation of cell migration and inflammation induced by these chemokines. This involvement is suggested by the observation that the deletion of the amino terminal 8 residues of MCP-1, amino terminal 9 residues of MCP-3, and amino terminal 8 residues of RANTES and the addition of a methionine to the amino terminus of RANTES, antagonize the chemotaxis, calcium mobilization and/or enzyme release stimulated by their native counterparts (Gong et al., 1996, J. Biol. Chem. 271:10521-10527; Proudfoot et al., 1996 J. Biol. Chem. 271:2599-2603). Additionally, xcex1 chemokine-like chemotactic activity has been introduced into MCP-1 via a double mutation of Tyr 28 and Arg 30 to leucine and valine, respectively, indicating that internal regions of this protein also play a role in regulating chemotactic activity (Beall et al., 1992, J. Biol. Chem. 267:3455-3459).
The monomeric forms of all chemokines characterized thus far share significant structural homology, although the quaternary structures of xcex1 and xcex2 groups are distinct. While the monomeric structures of the xcex2 and xcex1 chemokines are very similar, the dimeric structures of the two groups are completely different. An additional chemokine, lymphotactin, which has only one N terminal cysteine has also been identified and may represent an additional subgroup (xcex3) of chemokines (Yoshida et al., 1995, FEBS Lett. 360:155-159; and Kelner et al., 1994, Science 266:1395-1399).
Receptors for chemokines belong to the large family of G-protein coupled, 7 transmembrane domain receptors (GCR""s) (See, reviews by Horuk, R., 1994, Trends Pharmacol. Sci. 15:159-165; and Murphy, P. M., 1994, Annu. Rev. Immunol. 12:593-633). Competition binding and cross-desensitization studies have shown that chemokine receptors exhibit considerable promiscuity in ligand binding. Examples demonstrating the promiscuity among xcex2 chemokine receptors include: CCR-1, which binds RANTES and MIP-1xcex1 (Neote et al., 1993, Cell 72:415-425), CCR-4, which binds RANTES, MIP-1xcex1, and MCP-1 (Power et al., 1995, J. Biol. Chem. 270:19495-19500), and CCR-5, which binds RANTES, MIP-1xcex1, and MIP-1xcex2 (Alkhatib et al., 1996, Science, in press and Dragic et al., 1996, Nature 381:667-674). Erythrocytes possess a receptor (known as the Duffy antigen) which binds both xcex1 and xcex2 chemokines (Horuk et al., 1994, J. Biol. Chem. 269:17730-17733; Neote et al., 1994, Blood 84:44-52; and Neote et al., 1993, J. Biol. Chem. 268:12247-12249). Thus the sequence and structural homologies evident among chemokines and their receptors allows some overlap in receptor-ligand interactions.
CCR-5 is the major coreceptor for macrophage-tropic strains of HIV-1 (Alkhatib et al., 1996, Science 272:1955-1958; Choe et al., 1996, Cell 85:1135-1148; Deng et al., 1996, Nature 381:661-666; Doranz et al., 1996, Cell 85:1149-1158; Dragic et al., 1996, Nature 381:667-674). RANTES, MIP-1xcex1, or MIP-1xcex2, the chemokine ligands for this receptor have been shown to block HIV Env-mediated cell fusion directed by CCR-5 (Alkhatib et al., 1996, Science 272:1955-1958 and Dragic et al., 1996, Nature 381:667-674). Additional support for the role of CCR-5 as an M-tropic HIV-1 cofactor comes from the finding that a 32-base pair deletion in the CCR-5 gene found in three multiply exposed, but uninfected individuals, prevents HIV from infecting macrophages (Liu et al., 1996, Cell 86:367-377). However, only three of the 25 uninfected individuals studied had this mutation. This 32-base pair deletion in the CCR-5 gene is found in many (1% uninfected approximately 13% non-risk) caucasians. See also Huang et al., 1996, Nature Med. 2:1240-1243; Samson et al., 1996, Nature 382:722-725; Dean et al., 1996, Science 273:1856-1862; and Michael et al., 1997, Nature Med. 3:338-340.
CD4+ T lymphocytes from these individuals, moreover, are not infected by HIV-1 in vitro. A disproportionately high number of these individuals are homozygous for a mutant CC-CKR5 allele that contains a 32 base pair deletion (Dean et al., 1996, Science 273:1856-1862).
The V3 loop of gp120 is the major determinant of sensitivity to chemokine inhibition of infection or replication (Cocchi et al., 1996, Nature Medicine 2:1244-1247; and Oravecz et al., 1996, J. Immunol. 157:1329-1332). Signal transduction through xcex2 chemokine receptors is not required for inhibition of HIV infection or replication, since RANTES inhibits HIV-1 infection in the presence of pertussis toxin, an inhibitor of G-protein-mediated signaling pathways (P. M. Murphy 1994, Ann. Rev. Immunol. 12:593-633; Bischoff et al., 1993, Eur. J. Immunol. 23:761-767; and Simon et al., 1991, Science 252:802-807). In addition, mutant chemokine receptors that lack normal function retain the ability to act as HIV coreceptors. For example, deletion of xe2x80x9csignalling sequencesxe2x80x9d have no effect on coreceptor function (Lu et al., 1997, Proc. Natl. Acad. Sci. USA 94:6426) and mutant chimeras that do not signal chemotaxis do act as coreceptors (Atchison et al., 1996, Science 274:1924). CxC CKR4, a CxC (xcex1) chemokine receptor, has been shown to be a coreceptor involved in infection by laboratory-adapted HIV-1 strains (Fong et al., 1996, Science 272:872-877). The xcex1 chemokine SDF-1, the ligand for this receptor, has been demonstrated to block infection by T-tropic HIV-1 isolates. CxC CKR4 does not bind the beta chemokines RANTES, MIP-1xcex1, or MIP-1xcex2.
Recently, it has been shown that certain primary, syncytium-inducing/T-tropic isolates use both CC CKR5 and CxC CKR4 as coreceptors and are able to switch between the two. Thus, in the presence of RANTES, MIP-1xcex1 and MIP-1xcex2, the chemokine ligands for CCR-5, T-tropic strains are still able to infect cells via the CxC CKR4 coreceptor (Zhang et al., 1996, Nature 383:768).
Godiska et al. identified and described the nucleic acid and amino acid sequences of an additional xcex2 chemokine designated the macrophage derived chemokine (MDC) (PCT Publication WO 96/40923 dated Dec. 19, 1996, and 1997, J. Exp. Med. 185:1595-1604). The PCT publication WO 96/40923 further provides materials and methods for the recombinant production of the chemokine, the purified and isolated chemokine protein, and polypeptide analogues thereof. While the PCT publication WO 96/40923 discloses that a need exists for additional Cxe2x80x94C chemokines for use as inhibitors of strains of HIV (see page 4, lines 15-23) and discloses an assay for determining whether MDC has HIV inhibiting effects (see page 17, lines 16-17 and Example 20), the reference fails to teach or provide any evidence that MDC does inhibit HIV proliferation or what strains of HIV MDC would be effective against. There is no enabling disclosure therein of using MDC to treat or prevent HIV infection or disorders stemming therefrom.
Further, PCT Publication WO 96/40923 dated Dec. 19, 1996, states that the established correlation between chemokine expression and inflammatory conditions and disease states provides for diagnostic and prognostic uses for chemokines (see page 5). However, the PCT publication fails to disclose or suggest methods for diagnosis or prognosis of HIV infection using MDC expression. Both of the Godiska et al. references fail to detect MDC expression in unactivated or activated PBMCs.
Klotman et al. (May 4, 1997, Abstract page 40, Conference on Advances in AIDS Vaccine Development, 9th Annual Meeting of the National Cooperative Vaccine Development Group for AIDS) describes a method for producing an anti-HIV-1 factor from cultured, transformed CD8+ cell supernatants. A factor was purified by concentrating cell supernatants and subjecting them to size fractionation, ion exchange and reverse-phase chromatography. The factor is apparently distinct from RANTES, MIP-1xcex1 and MIP-1xcex2. No hysical characteristics (e.g., molecular weight, sequence, etc.) of the factor are disclosed.
HIV infection is pandemic and HIV-associated diseases represent a major world health problem. Although considerable effort is being put into the design of effective therapeutics, currently no curative anti-retroviral drugs against AIDS exist. In attempts to develop such drugs, several stages of the HIV life cycle have been considered as targets for therapeutic intervention (Mitsuya et al., 1991, FASEB J. 5:2369-2381). Many viral targets for intervention with the HIV life cycle have been suggested, as the prevailing view is that interference with a host cell protein would have deleterious side effects. For example, virally encoded reverse transcriptase has been one focus of drug development. A number of reverse-transcriptase-targeted drugs, including 2xe2x80x2, 3xe2x80x2-dideoxynucleoside analogues such as AZT, ddI, ddc, and d4T have been developed which have been shown to been active against HIV (Mitsuya et al., 1991, Science 249:1533-1544).
The new treatment regimens for HIV-1 show that a combination of anti-HIV compounds, which target reverse transcriptase (RT), such as azidothymidine (AZT), lamivudine (3TC), dideoxyinosine (ddi), dideoxycytidine (ddc) used in combination with an HIV-1 protease inhibitor have a far greater effect (2 to 3 logs reduction) on viral load compared to AZT alone (about 1 log reduction). For example, impressive results have recently been obtained with a combination of AZT, ddI, 3TC and ritonavir (Perelson et al., 1996, Science 15:1582-1586). However, it is likely that long-term use of combinations of these chemicals will lead to toxicity, especially to the bone marrow. Long-term cytotoxic therapy may also lead to suppression of CD8+ T cells, which are essential to the control of HIV, via killer cell activity (Blazevic et al., 1995, AIDS Res. Hum. Retroviruses 11:1335-1342) and by the release of factors which inhibit HIV infection or replication, notably the chemokines Rantes, MIP-1xcex1 and MIP-1xcex2 (Cocchi et al., 1995, Science 270:1811-1815). Another major concern in long-term chemical anti-retroviral therapy is the development of HIV mutations with partial or complete resistance (Lange, J. M., 1995, AIDS Res. Hum. Retroviruses 10: S77-82). It is thought that such mutations may be an inevitable consequence of anti-viral therapy. The pattern of disappearance of wild-type virus and appearance of mutant virus due to treatment, combined with coincidental decline in CD4+ T cell numbers strongly suggests that, at least with some compounds, the appearance of viral mutants is a major underlying factor in the failure of AIDS therapy.
Attempts are also being made to develop drugs which can inhibit viral entry into the cell, the earliest stage of HIV infection, by focusing on CD4, the cell surface receptor for HIV. Recombinant soluble CD4, for example, has been shown to inhibit infection of CD4+ T cells by some HIV-1 strains (Smith et al., 1987, Science 238:1704-1707). Certain primary HIV-1 isolates, however, are relatively less sensitive to inhibition by recombinant CD4 (Daar et al., 1990, Proc. Natl. Acad. Sci. USA 87:6574-6579). In addition, recombinant soluble CD4 clinical trials have produced inconclusive results (Schooley et al., 1990, Ann. Int. Med. 112:247-253; Kahn et al., 1990, Ann. Int. Med. 112:254-261; Yarchoan et al., 1989, Proc. Vth Int. Conf. on AIDS, p. 564, MCP 137).
The late stages of HIV replication, which involve crucial virus-specific processing of certain viral encoded proteins, have also been suggested as possible anti-HIV drug targets. Late stage processing is dependent on the activity of a viral protease, and drugs are being developed which inhibit this protease (Erickson, J., 1990, Science 249:527-533). The clinical outcome of these candidate drugs is still in question.
Attention is also being given to the development of vaccines for the treatment of HIV infection. The HIV-1 envelope proteins (gp160, gp120, gp41) have been shown to be the major antigens for neutralizing anti-HIV antibodies present in AIDS patients (Barin et al., 1985, Science 228:1094-1096). Thus far, therefore, these proteins seem to be the most promising candidates to act as antigens for anti-HIV vaccine development. Several groups have begun to use various portions of gp160, gp120, and/or gp41 as immunogenic targets for the host immune system. See, for example, Ivanoff et al., U.S. Pat. No. 5,141,867; Saith et al., WO 92/22654; Shafferman, A., WO 91/09872; Formoso et al., WO 90/07119. To this end, vaccines directed against HIV proteins are problematic in that the virus mutates rapidly rendering many of these vaccines ineffective. Clinical results concerning these candidate vaccines, however, still remain far in the future.
Thus, although a great deal of effort is being directed to the design and testing of anti-retroviral drugs, effective, non-toxic treatments are still needed.
Citation of a reference hereinabove shall not be construed as an admission that such reference is prior art to the present invention.
The present invention relates to prophylactic and therapeutic methods and compositions based on MDC proteins, nucleic acids, derivatives or analogues thereof that inhibit replication and/or infection of a lentivirus, particularly an immunodeficiency virus in vitro or in vivo, decrease viral load, and/or treating or preventing diseases and disorders associated with infection of a lentivirus, particularly an immunodeficiency virus. In specific embodiments, the lentivirus inhibited by the methods and compositions of the invention is HIV.
The present invention also relates to therapeutic compositions based on MDC and nucleic acids encoding MDC. Therapeutic compounds of the invention include, but are not limited to, MDC, nucleic acids encoding MDC, and derivatives (including, but not limited to, fragments and chimerics) and analogues thereof, that are effective at inhibiting replication or infection by an immunodeficiency virus.
The invention further relates to therapeutic methods for treatment and prevention of diseases and disorders associated with infection with a lentivirus, in particular an immunodeficiency virus, in particular HIV infection, by administering a therapeutic composition of the invention.
The invention further relates to methods for prognosis of lentivirus infection, in particular HIV infection, using MDC as a prognostic indicator.
The invention further provides MDC proteins, and nucleic acids encoding such proteins, that have amino-terminal sequences that differ from other molecular forms of MDC. Recombinant host cells and methods of production are also provided.